Abscisic acid (1, ABA) is a plant hormone involved in the regulation of important developmental functions including seed maturation, desiccation tolerance and dormancy, as well as adaptation to environmental stress through stomatal closure and modification of gene expression.1-3 The biosynthesis of ABA 1 begins with isopentenyl diphosphate which enters the mevalonic acid-independent 2-C-methyl-d-erythritol-4-phosphate pathway producing plastidic isoprenoids, including carotenoids.4 Enzymatic cleavage of C40 carotenoid cis-xanthophylls (neoxanthin 2 and violaxanthin 3) at the 11′-12′ double bond by a 9-cis-epoxycarotenoid dioxygenase (NCED) produces C15 (xanthoxin 4) and C25 metabolites and represents the first committed step in ABA biosynthesis (FIG. 1). Xanthoxin 4 is subsequently converted by an alcohol dehydrogenase (ABA2) into abscisyl aldehyde 5, which is oxidized to ABA 1 by an abscisic aldehyde oxidase (AAO3).3 The catabolism of ABA occurs principally through oxidation of one of the methyl groups of the ring (8′-carbon atom, using convention for ABA numbering) mediated by members of a class of P450 monooxygenase enzymes, CYP 707A.5 The catabolite phaseic acid (6, PA) which occurs as the result of reversible cyclization of 8′-hydroxyABA, is reduced by an unknown reductase to afford dihydrophaseic acid (7, DPA). ABA can also be metabolized to the glucose conjugate 8.3 
First identified in maize (VP14), NCEDs have also been found in a variety of other species including Arabidopsis thaliana (AtNCED3), bean (PvNCED1), tomato (LeNCED1), avocado (PaNCED1 and PaNCED3) and cowpea (VuNCED1).6-11 AtNCED3 is a member of the carotenoid cleavage enzyme family of Arabidopsis thaliana, which consists of nine enzymes.12 In general, the family is characterized by a plastid-targeting transit peptide, an amphipathic α-helix domain and a catalytic domain which contains four conserved histidine residues responsible for non-heme iron co-ordination. AtNCED3 is found in both the stroma and bound to the thylakoid membrane, accounts for NCED activity in roots, contributes to NCED activity in developing seeds and is the major stress-induced NCED in leaves of Arabidopsis thaliana.12 Recently, immunohistochemical analysis revealed that the AtNCED3 protein is detected exclusively in the vascular parenchyma cells of water-stressed plants.13 Due to ABA's important role in plant physiology, significant effort has been expended on investigating functional aspects of ABA 1 biosynthesis, regulation and action. ABA-deficient mutants are powerful tools for elucidating ABA's role in planta, as are chemical inhibitors of ABA 1 biosynthesis which have broad applicability to many plant species.
General carotenoid biosynthesis inhibitors such as fluridone, a potent broad spectrum herbicide that inhibit phytoene desaturase in the carotenoid biosynthesis pathway, have been used to inhibit ABA 1 biosynthesis.14,15 While fluridone does inhibit ABA 1 biosynthesis, a corresponding general repression of the carotenoid biosynthesis pathway limits its application for biochemical investigations including those of carotenoid cleavage enzymes and products. To address this problem, Abamine compounds 9 and 10 were developed as inhibitors of NCED's, based on early observations that a number of inhibitors of soybean lipoxygenase were effective in reducing ABA accumulation in stressed soybean cell cultures and seedlings.16 One of the active compounds, nordihydroguaiaretic acid, served as the starting structure for generation of analogs with improved NCED inhibitory activity, leading to development of the tertiary amines Abamine (9, ABM) and Abamine SG (10, ABM-SG) (FIG. 2).17,18 Arabidopsis plants treated with ABM 9 showed a significant decrease in drought tolerance and under simulated osmotic stress ABM 9 inhibited stomatal closure in spinach leaves. The latter effect was counteracted by co-application of ABA 1. ABM-SG 10 strongly inhibited the expression of ABA-responsive and catabolic genes in plants under osmotic stress. Finally, both ABM 9 and ABM-SG 10 reduced ABA metabolite accumulation by 35% and 77% respectively and were shown to act as competitive inhibitors of the cowpea NCED enzyme, with Ki's of 18.5 μM and 38.8 μM respectively.
There remains a need for NCED inhibitors for use in regulating ABA biosynthesis in plants.